Rapid Diagnosis of α-Thalassemia by Melting Curve Analysis

α-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of α-globin protein. Couples who both carry the α-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the α-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study, a new and accurate method for α-thalassemia diagnosis was developed by genotyping α-thalassemia-1, the Southeast Asian type (−−SEA) and Thai type (−−THAI) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments, the −−SEA and −−THAI deletions and two normal fragments, ψζ- and α2-globin gene. The primers were capable of distinguishing α-thalassemia 1 heterozygotes from α-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the −−THAI, −−SEA, ψζ-globin, and α2-globin gene fragments were 79.9 ± 0.2, 89.4 ± 0.5, 92.8 ± 0.2, and 85.0 ± 0.2°C, respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis, and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process, which is laborious, and allows high throughput screening suitable for large population screening for prevention and control of thalassemia.